DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHS)
IN SEAFOOD USING GC-MS: A COLLABORATIVE STUDY
Lucie Drábová, Jana Pulkrabová, Kateřina Maštovská, Kamila Kalachová, Vladimír Kocourek, Jana Hajšlová
5th Meeting on Chemistry and Life 201114‐16th September 2011, Brno, Czech Republic
AOAC CALL: PAHS IN FISH AND SEAFOOD
NEW METHOD FOR MULTIPLE POPs
AOAC: COLLABORATIVE STUDY
Talk Summary
5th Meeting on Chemistry and Life 2011
Oil Spill - Gulf Of Mexico 2010
5th Meeting on Chemistry and Life 2011
Call for Methods 23/7/2010
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Isolation10 min
Clean up30 min
Identification &quantification
1 h
BFR PCB PAH Non-ortho PCB
Extraction Shaking (H2O + ethylacetate)
Partition (transfer into organic phase)induced by MgSO4 + NaCl
Clean upSilicagel minicolumn
Identification & quantificationGC-QMS(EI)
GC-TOFMS (EI)GC×GC-TOFMS (EI)
6 SAMPLES / < 1 HOUR
SINGLE GC INJECTION
CONffIDENCEIntegrated sample preparation
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Target analytes
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Benz[a]anthracene BaABenzo[b]fluoranthene BbFABenzo[k]fluoranthene BkFABenzo[ghi]perylene BghiPBenzo[a]pyrene BaPChrysene CHRDibenz[a,h]anthracene DBahAIndeno[1,2,3-cd]pyrene IPBenzo[j]fluoranthene BjFABenzo[c]fluorene BcFLCyclopenta[cd]pyrene CPPDibenzo[a,e]pyrene DBaePDibenzo[a,h]pyrene DBahPDibenzo[a,i]pyrene DBaiPDibenzo[a,l]pyrene DBalP5-Methylchrysene 5-MC
15 + 1 EU PAHsAcenaphthene ACAcenaphthylene ACLAnthracene ANFluoranthene FAFluorene FLNaphthalene NAPhenanthrene PHEPyrene PY
16 US EPA PAHs
methylated homologues2-Methylanthracene 2-MA1-Methylchrysene 1-MC3-Methylchrysene 3-MC1-Methylnaphthalene 1-MN2-Methylnaphthalene 2-MN1-Methylphenanthrene 1-MPH1-Methylpyrene 1-MP1,7-Dimethylphenanthracene 1,7-DMP2,6-Dimethylnaphthalene 2,6-DMN
Benzo[e]pyrene BePDibenzothiophene DBT
Method Performance Characteristics – PAHs and their homologues (GC-TOFMS)
750 1000 1250 1500 1750 2000 2250 25000
10000
20000
30000
40000
50000
60000
Time (s)128 142 152 153 166 178 202 216 226 228 242 252 276 278302
Naph
1‐MN
ACL
ACFL
DBT
PHE
AN
FA PY
1‐MPH 2‐M
A
BaA
CPP
CHR
1‐MC
5‐MC
BbFA
BkFA
BjFA BaP DB
ahA
IP
BghiP
DBalP
DBaeP
DBaiP
DBah
P
2‐MN
3‐MC
SHRIMPSRecovery: 69 – 109 %Repeatability: 2 – 15 %LOQ: 0.05 – 0.25 µg.kg‐1
FISH Recovery: 75 – 107 %Repeatability: 2 – 13 %LOQ: 0.05 – 0.25 µg.kg‐1
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DB-EUPAH (20 m × 0.18 mm × 0.14 µm)
21/10/2010
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Target analytes – AOAC study1,7-Dimethylphenanthracene 1,7-DMP1-Methylnaphthalene 1-MN1-Methylphenanthrene 1-MPH2,6-Dimethylnaphthalene 2,6-DMN3-Methylchrysene 3-MCAnthracene ANBenz[a]anthracene BaABenzo[a]pyrene BaPBenzo[b]fluoranthene BbFABenzo[ghi]perylene BghiPBenzo[k]fluoranthene BkFAChrysene CHRDibenz[a,h]anthracene DBahAFluoranthene FAFluorene FLIndeno[1,2,3-cd]pyrene IPNaphthalene NAPhenanthrene PHEPyrene PY
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19 PAHs
AOAC Int. Collaborative StudyStudy phases:
(1) Laboratory qualification
(2) Test sample analysis
Study design:
3 matrices: mussel, oyster, shrimp total of 5 different levels of BaP (2 – 50 µg/kg) other studied PAHs at varying levels from 2 to 250 µg/kg
that mimic typical PAH patterns each matrix fortified at 3 different concentration levels in
duplicate + one blank for each matrix total of 7 x 3 = 21 study samples
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AOAC Int. Collaborative StudyStudy participants: Adpen Laboratories (FL, USA) Covance Laboratories (WI, USA) EU PAH Reference Laboratory (Belgium) Eurofins CAL (LA, USA) FL Dept. of Agriculture and Consumer Services (FL, USA) Food Safety Net Services (TX, USA) Institute of Chemical Technology (Czech Republic) LECO Corporation (MI, USA) MD Dept. of Agriculture (MD, USA) MI Dept. of Community Health (MI, USA) Microbac Laboratories (IN, USA) Premier Laboratory (CT, USA) Silliker JR Laboratories (BC, Canada) Thermo Fisher Scientific FSRC (Germany) State Veterinary Institute in Praha, (Czech Republic)
Study direction team: Co-study directors: K. Mastovska (Covance), W. Sorenson (Covance), and
J. Hajslova (ICT) Technical advisors: J. Schmitz (Covance), J. Pulkrabova (ICT)
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15 laboratories
(1) Laboratory qualification phase
Optimization of GC-MS, silica-SPE clean-up and solvent evaporation conditionsCheck of potential reagent blank contaminationFamiliarization with the method
Qualification steps:(1) GC separation test(2) Calibration range test(3) Solvent evaporation test(4) PAH and fat elution profiles(5) Procedure blank test(6) Low-level spike test(7) Practice sample analysis
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1. GC Separation Test
(1) a baseline separation of benzo(a)pyrene and benzo(e)pyrene(2) at least 50% valley separation of anthracene and phenanthrene(3) at least 50% valley separation for benzo(b)fluoranthene, benzo(j)fluoranthene,
and benzo(k)fluoranthene
To optimize GC separation of PAHs
(1) (2) (3)
BeP
BaP
Phe
AN
BbFBkFBjF
Criteria:
(1) DB‐EUPAH (20m x 0.18 mm x 0.14 µm) (Agilent J&W, USA)(2) Rxi‐17 Sil (30m x 0.25 mm x 0.25 µm) (Restek, USA)(3) DB‐17 MS (30m x 0.25 mm x 0.25 µm) (Agilent J&W, USA)(4) TR‐50MS (30m x 0.25 mm x 0.25 µm) (Thermo Fisher Scientific, USA)(5) ZB‐50 (30m x 0.25 mm x 0.25 µm) (Phenomenex, USA)
GC columns used in the study
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y = 0.0181x + 0.0937R² = 0.9999
02468
101214161820
0 200 400 600 800 1000Nor
mal
ized
Res
pons
e
Concentration (µg/L)
BaP
y = 0.0215x + 0.1677R² = 1
0
20
40
60
80
100
120
0 1000 2000 3000 4000 5000Nor
mal
ized
Res
pons
e
Concentration (µg/L)
Naph
2. Calibration Range Test
8-point calibration curves Calibration range: 5 –1000 µg/L for BaP and lower levels PAH, 12.5 – 2500 µg/L
for higher-level PAHs, 25 –5000 µg/L for naphthalene Coefficients of determination (r2) of 0.990 or greater are required Test for carry-over (response in the solvent blank < 0.5% of the highest standard)
To determine linear range for analyte responses normalized to respective 13C-PAHs, test carry-over, and optimize injection conditions
r2: 0.9987 – 1.000Max residuals ± 20% 5th Meeting on Chemistry and Life 2011
3. Solvent Evaporation TestTo determine absolute recoveries of PAHs and 13C-PAHs during two evaporation experiments simulating the two evaporation steps in the method
The absolute recoveries - above 70% Recoveries of the heavier PAHs - in the range of 90-110%
(a) evaporation of 5 mL of an PAH/13C-PAH solution in EtOAc and reconstitution in isooctane
(b) evaporation of 10 mL of an PAH/13C-PAH solution in hexane:DCM (3:1, v/v) and reconstitution in isooctane Criteria:
Evaporation techniques employed in the study: nitrogen blown-down rotary vacuum evaporation
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Recommendations: use isooctane as a keeper in both evaporation steps add 1-2 mL of EtOAc prior to the second evaporation step to improve recoveries of volatile PAHs
Recoveries : 78 – 109 %
4. PAH and Fat Elution Profiles
0102030405060708090
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0% Ana
lyte eluted
Elution volume (mL)
LIPIDS
0102030405060708090
100
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
% Ana
lyte eluted
Elution volume (mL)
Commercial SPE silicacartridge (UCT)
100 mg of fat loaded on column containing 1G of
sorbent
100 mg of fat loaded on column containing 1G of
sorbent
in‐house prepared mini ‐
column
LIPIDS
To determine elution profiles of PAHs and fat on the silica gel SPE cartridge of your choice and optimize the elution solvent volume
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5. Procedure Blank TestTo evaluate and minimize background contamination, potentially occurring from different sources
Potential contamination sources:
Laboratory air
Solvents
Salts (have to be muffled)
Glassware
Extraction tubes (certain PAHs released from contaminated tubes when heated by the exothermic reaction caused by addition of anhydrous MgSO4 to the aqueous extract)
Criteria: Concentration of PAHs in blank < lowest calibration level Concentration of Naph < 50 µg/mL (equivalent to 5 µg/g sample)
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6. Low-Level Spike TestAnalyze 7 replicates of a blank shrimp matrix spiked at PAH concentrations
equivalent to the fitness-for-purpose LOQ for BaP of 1 μg/kg and corresponding
concentrations of other PAHs (based on their concentration ratio in the
calibration solutions)
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Average recovery (%, n = 7)PAH Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 Lab 7 Mean RSD (%)1-MN 167 127 124 106 110 83 95 116 23
Ant 105 98 94 84 93 96 91 94 6
BaP 103 85 89 82 92 98 85 90 8
BghiP 109 111 88 80 92 106 82 95 14
Recoveries : 90 – 139 %Repeatability: 8 – 23 %
Familiarization with the complete analysis procedure. This will ensure that instrumentation and reagents are all working properly in advance of conducting the actual study
Three samples of two varying matrices Sample of NIST SRM 1974b - Organics in Mussel Tissue (Mytilus edulis)
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7. Practice Samples
NIST SRM 1974b - Recoveries (%)PAH µg/kg Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 Lab 7 Lab 8 Mean RSD (%)BaA 4.74 90 83 80 86 73 93 85 89 85 7BaP 2.8 99 80 79 76 72 79 76 89 81 11BghiP 3.12 103 99 93 96 84 100 101 109 98 8Flt 17.1 104 102 93 102 85 100 103 103 99 7
Two samples of spiked shrimpsSHRIMP Practice Sample - Recoveries (%)
PAH Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 Lab 7 Mean RSD (%)1-MN 81 82 88 122 77 57 146 93 33Ant 63 71 75 89 66 58 59 69 16BaP 61 71 63 74 57 38 51 59 20BghiP 60 74 62 72 57 39 50 59 21
Low-Level Spike Test and Practice samplesRecovery %
PAH low level spikes practice samples-spiked shrimps
1,7-DMP 84 481-MN 103 931-MP 92 622,6-DMN 96 483-MC 81 61Ant 105 69BaA 101 56BaP 103 59BbF 104 56BghiP 109 59BkF 101 58Chr 105 56DBahA 102 57Fln 91 68Flt 107 58IcdP 102 57Naph 113 85Phe 92 65Pyr 107 59
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Extraction time
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0
50
100
150
200
250
300
1 min 15 min 1 min 15 min 1 min 15 min 1 min 15 min
Unpeeled shrimp Peeled shrimp Unpeeled shrimp Peeled shrimp
LN2‐homogenized processed by blender only
Recovery %
1‐MN Ant BaP BghiP
The influence of sample preparation on recovery of PAHs in spiked shrimps
Recoveries around 100% were obtained using the original method for all tested PAHs when both peeled and unpeeled shrimp were processed by blender only
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0
20
40
60
80
100
120
1 h 2 days 5 days 2 days 5 days
RT (20°C) Freezer (‐20°C) Refrigerator (4°C)
Recovery %
1‐MN Ant BaP BghiP
The influence of storage temperature on recovery of PAHs in spiked shrimps
Storage temperature plays an important role. Recoveries for fortified shrimp stored in a refrigerator dropped to about 40-50%.
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0
30
60
90
120
1 min 5 min 15 min 30 min 60 min
Recovery %
1‐MN Ant BaP BghiP
The influence of extraction time on recovery of PAHs in spiked shrimps
Spiked peeled and blended shrimps
No significant differences were observed in analyte recoveries using the different extraction times.
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0
50
100
150
200
250
5 mL water 10 mL water 15 mL water 5 mL water
Sample at room temperature Frozen sample
Recovery
%
1‐MN Ant BaP BghiP
The influence of water amount on recovery of PAHs in spiked shrimps
No differences in PAH recoveries were observed because even the recoveries obtained with 5 mL addition in thawed samples were high
Shrimp represent a more viscous matrix, for which a larger volume (10 mL) addition could improve shaking and matrix-solvent interaction
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Optimized shrimp samples preparationPAH Mean RSD (%)
1,7-DMP 99 121-MN 95 121-MP 94 112,6-DMN 94 123-MC 101 11Ant 90 12BaA 92 10BaP 92 9BbF 92 9BghiP 88 8BkF 95 10Chr 94 10DBahA 93 10Fln 91 11Flt 92 11IcdP 91 11Naph 97 13Phe 91 10Pyr 92 11
processed by blender only spiked before extraction in
laboratory 10 ml of water used for extraction 1 min of shaking for extraction Sample store temperature ≤ - 20°C
Recoveries : 88 – 101 %Repeatability: 8 – 13 %
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(2) Test sample analysis 3 matrices: mussels, oysters, shrimps
total of 5 different levels of BaP (2 - 50 µg/kg)
other studied PAHs at varying levels from 2 to 250 µg/kg that mimic typical PAH patterns each matrix fortified at 3 different concentration levels in duplicate
Preliminary results:
n Recovery % RSD %
Mussels 6 82–111 1–6Oysters 6 48–106 2–13 Shrimps 6 78–117 3–10
Lower recoveries of BaP and Ant from oyster samples stored at -20°C
Conclusions
15 laboratories started the collaboration study
The problems with the recovery of the spiked shrimps were resolved
10 laboratories have been qualified and started theactual study (analysis of the test samples)
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(1) Laboratory qualification
(2) Test sample analysis Preliminary results:
Mussels, oysters and shrimps:Recovery: 48 – 117%Repeatability: 1 – 13%
Lower recoveries of BaP and Ant from oyster samplesstored at -20°C
Thank you for your attention…